Squamous cell carcinoma of the head and neck (HNSCC) is the 6th most common cancer in the developed world, affecting nearly 44,000 patients each year in the US, which results in ~11,000 deaths. The vast majority of these malignancies involve neoplastic lesions in the oral cavity, lip, and pharynx. Like most cancers, HNSCC results from a series of discrete, irreversible and sequential alterations in genes that control cell growth and differentiation, together with genetic aberrations promoting invasion and metastasis. The goal of our program is scientific excellence in addressing the devastating problem of oral cancer. In particular, we aim to elucidate the molecular changes that contribute to the evolution of oral neoplasia, and to use this knowledge to develop markers of disease progression and novel therapeutic approaches for oral malignancies. [unreadable] [unreadable] Genomic and proteomic approaches to understand oral cancer [unreadable] [unreadable] Although risk factors for HNSCC, such as alcohol and tobacco consumption, are well recognized, the molecular mechanisms responsible for this malignancy are still not fully understood. In this regard, our laboratory has used a number of novel approaches to investigate gene and protein expression profiles in HNSCC. We have been able to combine laser capture microdissection (LCM) with novel liquid protein extraction techniques and mass spectrometry to identify proteins expressed in normal oral squamous epithelium and HNSCCs displaying distinct differentiation patterns. Approximately 20,000 cells were procured from formalin-fixed paraffin embedded tissue sections of clinically defined cases of well, moderately, and poorly differentiated squamous cell carcinoma and normal epithelial cells. After LCM and protein extraction, the resulting tryptic peptides were analyzed by nanoflow reversed-phase liquid chromatography coupled with a linear ion-trap mass spectrometer operating in a data-dependent tandem mass spectrometry mode. Multiple separate LC-MS/MS analyses identified up to 900 unique proteins within each individual sample. We expect that the ability to correlate histopathological classification of disease with protein expression profiles will allow the development of true biomarkers of diagnostic and prognostic value, as well as help identify proteins whose expression or activity contribute to tumor progression.[unreadable] [unreadable] The genomic and proteomic analysis of HNSCC is already providing essential information about molecules uniquely expressed in cancer lesions. We have now initiated an effort aimed at developing rapid and sensitive methods for the detection of premalignant lesions or the presence of micrometastasis in sentinel lymph nodes. For this work, we have teamed up with Jim Rusling at UCONN to develop a nanotube-based immunodetection system. The combination of single-wall carbon nanotube forest platforms for amperometric immunosensors with multi-label secondary antibody-HRP-nanotube bioconjugates led to highly sensitive detection of biomarkers in serum and tissue lysates. These easily fabricated nano-immunosensors show excellent promise for clinical screening of cancer biomarkers and point-of-care diagnostics of HNSCC patients. [unreadable] [unreadable] Dysregulated signaling networks in HNSCC: novel mechanism-based approaches for HSNCC treatment [unreadable] [unreadable] Recent findings suggest that the ability of Akt to coordinate mitogenic signaling with nutrient-sensing pathways controlling protein synthesis may represent an essential mechanism whereby Akt ultimately regulates cell growth. This pathway is initiated by Akt phosphorylation and inactivation of a tumor-suppressor protein, tuberous sclerosis complex protein 2 (TSC2), which is also known as tuberin. TSC2 associates with a second tumor-suppressor protein, tuberous sclerosis complex protein 1 (TSC1), and act together as a GTPase activating protein (GAP) for the small GTPase Rheb1. Thus, inactivation of TSC2 by Akt leads to the accumulation of the GTP-bound (active) form of Rheb1, which in turn promotes the phosphorylation and activation of an atypical serine/threonine kinase known as the mammalian target of rapamycin (mTOR). mTOR then phosphorylates key eukaryotic translation regulators, including p70-S6 kinase (p70S6K) and the eukaryotic translation initiation factor 4 E binding protein 1 (4E-BP1). The latter prevents the repressing activity of 4E-BP1 on the eukaryotic initiation factor 4E (eIF4E), ultimately resulting in enhanced translation from a subset of genes that are required for cell growth. Of direct relevance to HNSCC, eIF4E gene amplification and protein overexpression is often associated with malignant progression of this cancer type, and its expression levels in surgical margins can predict tumor recurrence. In a recent study, we showed that the Akt-mTOR pathway plays a central role in HNSCC. Concomitantly, we observed that rapamycin exerts a potent anti-tumor effect in vivo, as it inhibits cell proliferation and induces apoptotic cell death of HNSCC cells ultimately promoting tumor regression. These findings identified the Akt-mTOR pathway as a potential therapeutic target for HNSCC, thus raising the possibility of exploring the clinical activity of rapamycin and its analogs in HNSCC patients.[unreadable] [unreadable] Whereas activation of the EGFR is a frequent event in HNSCC, EGFR-independent mechanisms also contribute to the activation of key intracellular signaling routes, including signal-transducer-and-activator-of-transcription-3 (STAT3), NFkappaB, and Akt. Indeed, we have previously shown that the autocrine activation of the gp130 cytokine receptor in HNSCC cells by tumor-released cytokines, such as IL-6, can result in the EGFR-independent activation of STAT3. In a recent study study, we explored the nature of the molecular mechanism underlying the enhanced IL-6 secretion in HNSCC cells. We found that HNSCC cells display increased activity of the IL-6 promoter, which was dependent on the presence of an intact NFkappaB site. Furthermore, NFkappaB inhibition downregulated IL-6 gene and protein expression, and decreased the released of multiple cytokines. Interestingly, interfering with NF?UB function also prevented the autocrine/paracrine activation of STAT3 in HNSCC cells. These findings provided the first evidence of a cross-talk between the NFkappaB and STAT3 signaling system, and support the emerging notion that HNSCC results from the aberrant activity of a signaling network rather than from the activation of a single signaling cascade.[unreadable] [unreadable] Animal models for oral malignancies [unreadable] [unreadable] We have recently studied in detail the biological consequences of the conditional expression of the ras oncogene in salivary glands in adult mice. We observed that the inducible expression of a mutated K-ras gene under the control of the K5 promoter in the salivary glands leads to the development of a complex array of preneoplastic epithelial lesions and carcinomas, with an incidence of 100% of the animals, and a minimum latency of as little as one week. All major salivary glands were affected, as well as a set of previously undescribed buccal accessory salivary glands anatomically located on the apex of the masseter muscle, close to the oral angle. The tumors appear to arise from the K5-positive basal cell compartment. Myoepithelial cells participate of the hyperplastic process but not in carcinomas, as the tumors are negative for smooth muscle actin. Carcinomas do not accumulate immunoreactive p53, but are strongly positive for a splice variant of p63 that can inhibit p53 transcriptional activity. This study provided evidence that the ras oncogene, when targeted to a specifically sensitive cell compartment within the salivary glands, can trigger a series of event that are sufficient for full carcinogenesis.